EP1134586A1

EP1134586A1 - Method for adding a fluid in a series of wells

Info

Publication number: EP1134586A1
Application number: EP00200813A
Authority: EP
Inventors: Werner René Irène De Beukeleer; Christiaan Hubert Simon Roelant
Original assignee: Tibotec BVBA
Current assignee: Janssen Infectious Diseases Diagnostics BVBA
Priority date: 2000-03-08
Filing date: 2000-03-08
Publication date: 2001-09-19
Also published as: NO20024274D0; AU3929701A; EP1264186A1; AU781327B2; US20030155034A1; AU781327C; NO20024274L; JP2003526498A; CA2400914A1; WO2001067114A1; NZ520524A; TW558639B; US6868875B2

Abstract

The present invention relates to a method for adding a predetermined quantity of fluid from a reservoir, through a fluid outlet in fluid communication with said reservoir, into each well of at least one series of wells.

Description

The present invention relates to a method for adding a predetermined quantity of fluid from a reservoir, through a fluid outlet in fluid communication with said reservoir, into each well of at least one series of wells.
Although the series of wells may consist of a series of separate wells positioned next to one another, the series of wells is usually comprised in multi-well or so-called microtiter plates. Such multi-well plates are currently used to perform a number of different applications, such as the screening of compounds in drug discovery programs, cell-based assays, synthesis reactions in combinatorial chemistry, polymerase chain reactions, etc. At present, dosing of fluids in the wells of multi-well plates requires quantities of the fluid to be taken up in one or more pipettes, such as piëzo-electric pipettes, and their release into the wells of said multi-well plates. Alternatively, metered quantities of the fluid are each time pumped through a nozzle in the successive wells. In art-known methods, the pipettes or nozzles need to be positioned first on top of or into the wells of the multi-well plate. Next, the fluid is ejected from the pipettes or nozzles. Subsequently, these pipettes or nozzles need to be positioned above or in neighbouring wells. As a result, a major drawback of these methods known in the art is that they are only suited for the handling of multi-well plates with a limited number of wells (in particular 96-well standard microtiter plates) but not for multi-well plates having a larger number of wells, such as, for example, 384, 864, 1536, 3456, 3840 or 9600 wells. Indeed, filling such a number of wells using art-known methods takes too much time and is therefore inefficient.
It is an object of a first aspect of the present invention to provide a new method that enables the addition of a predetermined quantity of a fluid into each well of a series of wells at high speed.
The method according to the invention is characterised in that, for adding said predetermined quantity of fluid into each well of said series of wells, said fluid outlet is moved relatively to said series of wells so that it passes in a continuous movement over said series of wells and during this passing said predetermined quantity of fluid is dispensed into each well of said series of wells.
According to the invention it has been found that the speed at which the wells of a multi-well plate can be filled is limited by the mechanical movement of the fluid outlet over the series of wells. In the prior art methods, this movement needs to be stopped each time when passing over one well. In the method according to the first aspect of the present invention, the fluid outlet is now passed in a continuous movement over the series of wells. The velocity at which the outlet may pass over the series of wells is therefore no longer limited by the limitations imposed by a discontinuous movement of the fluid outlet.
In the method according to the first aspect of the invention, dispensing fluid out of the fluid outlet may be stopped each time when passing from one well to another. In practice, such an interruption of the flow of fluid out of the fluid outlet generally does not impose a limit on the travelling speed of the fluid outlet since valves with very short opening times are readily available. Use can for example be made of diaphragm valves having opening times in the order of magnitude of some milliseconds or of so-called piëzo-electric valves which may have opening times as short as a few microseconds.
In a preferred embodiment of the method according to the first aspect of the invention, an uninterrupted flow of said fluid is however dispensed out of the fluid outlet when this outlet is passed over a series of wells.
Indeed, it has been found that the required quantities of fluid can also be obtained in the different wells without interrupting the flow of fluid out of the fluid outlet. An advantage of this preferred embodiment is clearly that a less complicated dispensing system is required since the flow of fluid has no longer to be interrupted at a rather high frequency and since no control system is required to synchronise the interruptions of the fluid flow with the movement over the series of wells.
According to a second aspect of the invention, it has moreover been found that dispensing very small amounts of fluid in the different wells by pulsed flow of this fluid may have deleterious effects on the fluid, in particular when this fluid contains biological material such as living cells or micro-organisms or macro-molecules.
In the method according to the second aspect of the present invention, a solution is provided to this problem which consists in that for adding said predetermined quantity of fluid into each well of said series of wells, said fluid outlet is moved relatively to said series of wells so that it passes over said series of wells and during this passing an uninterrupted flow of said fluid is dispensed out of the fluid outlet.
As set forth already above, the required quantities of fluid can indeed also be obtained in the different wells without interrupting the flow of fluid out of the fluid outlet. In this way, there is a smaller risk of damaging biological or other sensitive material which may be contained in the fluid. Although in this second aspect of the invention preference is also given to a continuous movement of the fluid outlet relative to the series of wells, one could possibly stop the movement of the outlet above each wells in particular when the fluid is dispensed at a small flow rate and a relatively large quantity of fluid is to be added into each well.
In a preferred embodiment of the methods according to both aspects of the invention, the fluid is dispensed uninterruptedly at a substantially constant flow rate out of said outlet when it passes over said series of wells. Preferably, the fluid outlet is moved at a substantially constant speed relative to the series of wells when it passes thereover. In this way, a substantially constant quantity of fluid is achieved in the different wells. In some cases, the travelling speed of the outlet and/or the flow rate of the fluid can however be increased or decreased, either continuously or discontinuously, when the outlet passes over the series of wells so that the quantity of fluid introduced therein increases or decreases in a corresponding way. Also when passing over different series of wells, the relative travelling speed of the fluid outlet and/or the flow rate of the liquid may be different for one series compared to another.
Other particularities and advantages of the invention will become apparent from the following description of some particular embodiments of the present method for adding predetermined quantities of fluid into at least one series of wells. The reference numerals used in this description relate to the annexed drawings wherein:
Figure 1 shows a perspective view of a standard 1536 well plate;
Figure 2 shows a pattern that may be followed by the liquid outlet to fill the 1536 well plate of Figure 1;
Figure 3 shows an alternative pattern which may be followed to fill the 1536 well plate when use is made of two liquid outlets in a fixed position with respect to one another; and
Figure 4 shows schematically a perspective view of an apparatus that can be used for introducing the fluid in the wells of a multi-well plate.
The present invention relates to a new method for adding a predetermined quantity of fluid into the wells 1 of a multi-well plate 2 or more generally in a series of wellsl, for example in one or more rows of a multi-well plate. The plate illustrated in Figure 1 comprises 1536 wells (32 x 48) and has a standard length L of about 128 mm and a standard width W of about 86 mm. The array of wells itself has a length 1 of about 109 mm and a width w of about 73 mm. The area of this array is thus smaller than 100 cm². Instead of 1536 wells, the plate of these standard dimensions may contain another multiplicity of the standard 96 wells such as, for example, 384, 864, 3456, 3840, 6144 or 9600 wells.
The invention is not limited to the nature of the fluid to be added into the wells. This fluid may for example be a liquid or a so-called semi-solid medium.
In case the wells are used with the intention to screen analytes, they are first filled with said analytes for which various techniques may be used. Subsequently, a fluid containing a so-called target is to be applied into the wells to test whether there is any analyte-target interaction. The target in particular may be composed of cells, viruses, molecules, receptors, beads or combinations thereof. One additional advantage of the second aspect of the present invention when using it for screening purposes is that there is little or no risk of harming living biological organisms, such as cells, while transferring said living organisms from the reservoir, which is in fluid communication with the outlet, to the wells in an uninterrupted flow.
In case synthesis reactions in combinatorial chemistry are performed in the series of wells, the fluid to be added may contain for instance a reagent in a particular solvent.
In an advantageous embodiment of the method according to the invention, the fluid is no longer separately added into the individual wells 1 by means of pipettes or by means of a pump which pumps each time a metered dose in the wells. Instead, the fluid is introduced in a series of wells 1 by dispensing an uninterrupted flow of the fluid out of a fluid outlet 3 and by simultaneously passing this fluid outlet 3, in a continuous movement, over the series of wells 1. Obviously, when having passed over the series of wells 1, the fluid flow may be interrupted. It has been found that notwithstanding the fact that the movement of fluid outlet 3 is not stopped above each of the wells and the fluid flow is not interrupted when moving to a next well, this method still allows to achieve the required quantities of fluid in each of the wells. Compared to existing discontinuous dosing techniques, this method offers the advantage that a larger amount of wells can be filled with the required quantity of liquid in a much shorter period of time. This advantage can even be achieved when the flow of fluid is each time interrupted when passing from one well to another since in practice high frequency valve mechanisms are readily available.
Before describing the further particularities of the method according to the invention, a short description will now be given of an apparatus that is suitable for performing this method.
The apparatus illustrated in Figure 4 comprises a frame 4 supported by four legs 5. On top of the frame 4, two reservoirs 6, 7 are provided each containing a fluid to be introduced in the wells 1 of a multi-well plate. The reservoirs 6, 7 are connected on the one hand to a source of pressurised gas 8 and on the other hand, through flexible tubings 9, 10 to dispense needles 11, 12. These dispense needles 11, 12 comprise a valve, for example a so-called diaphragm valve or a piëze-electric valve, for controlling the flow of fluid through the needles. As valve, use can for example be made of the 750 V series of general purpose diaphragm valves of EFD.
The bottom of these valves is provided with a tip adapter onto which tips 13, 14 of different sizes and showing in particular a different outlet 3, can be mounted. For controlling the operation of the dispense needles 11, 12, these needles are connected to a valve controller through the intermediary of pneumatic tubings 15, 16. Since the valve controller and the dispense needles are known per se, no further description will be given thereof.
In the apparatus illustrated in Figure 4, the needles 11, 12 are mounted on a carriage 17 which is movable up and downwards, according to double arrow 18 (Z-direction) on a guiding support 19 by means of an electric motor 20. The multi-well plate which is to be filed with the liquid is to be inserted in a plate holder 21 situated underneath the needles 11, 12 and showing a recess 22 with dimensions adapted to the outer dimensions of the standard multi-well plates 2. The plate holder 21 is slideably mounted on a carriage 23 so that it can be moved, according to a reciprocating movement, in the direction of double arrow 24 (X-direction) on the carriage 23 by means of an electric motor 25 which is also mounted on this carriage 23. In order to be able to move the plate holder 21 in two dimensions, the carriage 23 is slideably mounted, in the direction of double arrow 26 (Y-direction), onto a guiding support 27. This guiding support 27 is mounted within the frame and is provided with an electric motor 28 for moving the carriage 23, and hence the plate holder 21, in the Y-direction.
The apparatus shown in Figure 4 comprises a programmable control unit for controlling the operation of the valve controller and the operation of the motors 20, 25 and 28, i.e. the movements of the dispense needles 11, 12 in the Z-direction and the movements of the plate holder 21 in the X- and Y-direction. Further details of the control unit and the construction of the apparatus in general will not be given since a person skilled in the art will readily be able to construct such an apparatus and since many modifications can be applied to the illustrated apparatus without leaving the scope of the present invention.
Turning now back to the method according to the preferred embodiment of the invention wherein a series of wells 1 of a multi-well plate 2 is filled with fluid from a reservoir 6 or 7 by passing the fluid outlet 3 of the needles 13 or 14 in a continuous movement over this series of wells 1 while dispensing an uninterrupted flow of fluid out of the outlet, it will be clear that only a relative movement of the outlet 3 with respect of the multi-well plate 2 is required and that in other words either the outlet 3 and/or, as in the apparatus illustrated in Figure 4, the plate 2 are moved.
For adding the fluid into the wells 1 of the plate 2 illustrated in Figure 1, the different wells thereof could be considered as one series of wells which can be filled by one continuous movement of the outlet over the multi-well plate according to a pattern going from one well to the other and covering the entire surface of this plate. In this case, the movement direction of the outlet has however to be changed above certain wells so that very sudden changes of direction are required in order to avoid that the "corner" wells receive too much fluid (unless for example the wells on two opposite sides of the well may contain more fluid or unless the flow of fluid is interrupted when passing from one well to another).
In a preferred embodiment of the invention, the array of wells 1 of the multi-well plate 2 is therefore divided in several substantially parallel series of wells, in particular rows, extending from one side of the array to the other. In that case a pattern as illustrated in Figure 2 can be followed. According to this pattern, when having passed over one row, the fluid dispense outlet 3 turns in an area outside the array of wells 1 to another row of wells. In this way, the quantity of fluid introduced in the wells at both opposite ends of the plate is easier to control. Moreover, a less sharp turning has to be made when returning not in the next row but, as illustrated in Figure 2, by skipping a number of rows and by filling these rows during the next turns.
When passing over the area outside the array of wells, the flow of fluid out of the fluid outlet is preferably interrupted. Before passing over a next series of wells, the flow of fluid is started again, preferably somewhat in advance so as to fill the first well of each series at the required flow rate.
As described hereinabove, the apparatus illustrated in Figure 4 comprises two dispense needles 11 and 12 with a fluid outlet 3 and two fluid reservoirs 6, 7. These reservoirs 6, 7 may be filled with the same or with different fluids. When filled with the same fluid, it is of course possible to connect both outlets 3 to one and the same reservoir.
Both fluid outlets are fixed in the carriage 17 on a fixed distance from each other. This distance may correspond for example to half the number of rows of the multi-well plate. In this way, the plate can be filled according to a pattern as illustrated in Figure 3, whereby each outlet follows, on one half of the plate, a pattern similar to the pattern illustrated in Figure 2. It will be clear that with more outlets, similar patterns can be followed.
In a preferred embodiment, when the outlet 3 passes over one of said series of wells 1, the fluid is dispensed at a substantially constant flow rate out of the outlet. When the fluid outlet moves at a substantially constant speed with respect to the multi-well plate when it passes over this series of wells, a substantially constant quantity of fluid is dispensed in the different wells of this series.
If desired, the speed of the outlet with respect to the multi-well plate can either continuously or discontinuously be increased or decreased when passing over a series of wells. In this way, the quantity of fluid introduced in the wells will increase or decrease accordingly.
The flow rate at which the fluid is dispensed out of the outlet may for example be comprised between 0.001 and 2000 µl/sec and preferably between 10 and 1000 µl/sec and more preferably between 200 and 500 µl/sec. The quantity of fluid which may be added into each well may vary within wide ranges, in particular between 0.00025 and 100 µl. In case the flow rate comprises for example 350 µl/sec, one row of a 1536 well plate (32 x 48 wells) would for example be filled with 7 µl per well in about 1 sec. Filling the entire plate would take then about half a minute. Filling a row of a plate with a larger number of wells may require substantially the same time since each of the wells has normally to be filled with less fluid. Filling a 3456 well plate (48 x 72 wells) would take then about 50 sec, or even less in case two or more dispense outlets are used.
The above flow rates enable the use of very narrow needle tips or outlet openings. The outlets may have for example an inner diameter of between 40 and 500 µm, and more particularly of between 50 and 200 µm, at least in case the fluid has a viscosity comparable to the viscosity of water. It will be clear that such narrow jets of fluid will enable to fill multi-well plates having even up to 9600 wells (80 x 120 wells) on the standard area of about 109 x 73 mm. In this way, even multi-well plates having such an extremely high number of wells could be filled with fluid in an acceptable period of time by employing the method according to the invention.
In the method according to the second aspect of the invention, the relatively narrow flow of fluid dispensed out of the outlet is partially deposited onto the top surfaces of the walls separating the wells. It has been found that this quantity of fluid is however substantially equally divided over two successive wells when these wells are adjacent to one another. Preferably, the top surface of the partition walls between the wells is repellent for the fluid which is to be introduced therein so that small droplets are formed which roll off this surface so that no fluid remains on top thereof. Making this top surface somewhat convex, pointed or inclined can of course enhance this phenomenon. In the area where the fluid outlet or in other words the flow of fluid passes, the top surfaces of the partition walls between the wells have preferably a width smaller than 1 mm, and in particular smaller than 0.6 mm. Depending on the surface tension of the fluid and the adherence between the fluid and the surface of the partition walls, substantially no fluid will remain on top of such walls, even if these walls have no convex but a flat top surface.
From the above description, it will be clear that many modifications can be applied to the described embodiments without leaving the scope of the annexed claims.
It is for example possible to provide a fluid reservoir in the dispense needless 11, 12 themselves so that the tubings 9 and 10 can be omitted. Further, instead of moving the plate holder 21, it is also possible to move the dispense needles 11, 12 or to move the dispense needles 11,12 in one direction and the plate holder 21 in another direction.
The plate holder 21 may also be arranged to carry two or more multi-well plates. In this case, the dispense needles are preferably arranged on such a mutual distance that they move each simultaneously above one of the multi-well plates.
Finally, instead of using valves in the dispense needles and a pressurized source of fluid, a pump could be provided in the tubings leading from the reservoirs 7, 8 to the dispense needles.

Claims

A method for adding a predetermined quantity of fluid from a reservoir, through a fluid outlet in fluid communication with said reservoir, into each well of at least one series of wells, characterised in that, for adding said predetermined quantity of fluid into each well of said series of wells, said fluid outlet is moved relatively to said series of wells so that it passes in a continuous movement over said series of wells and during this passing said predetermined quantity of fluid is dispensed into each well of said series of wells.
A method according to claim 1, characterised in that during said passing an uninterrupted flow of said fluid is dispensed out of the fluid outlet.
A method for adding a predetermined quantity of fluid from a reservoir, through a fluid outlet in fluid communication with said reservoir, into each well of at least one series of wells, characterised in that for adding said predetermined quantity of fluid into each well of said series of wells, said fluid outlet is moved relatively to said series of wells so that it passes over said series of wells and during this passing an uninterrupted flow of said fluid is dispensed out of the fluid outlet.
A method according to any one of claims 1 or 3, characterised in that during said passing over said series of wells, the fluid is dispensed at a substantially constant flow rate out of said outlet.
A method according to any one of claims 1 to 4, characterised in that said fluid is dispensed at a flow rate of between 0.001 and 2000 µl/sec and preferably at a flow rate of between 10 and 1000 µl/sec and more preferably between 200 and 500 µl/sec.
A method according to any one of the claims 1 to 5, characterised in that said outlet has an inner diameter of between 40 and 500 µm, in particular between 50 and 200 µm.
A method according to any one of the claims 1 to 6, characterised in that the fluid outlet is moved at a substantially constant relative speed with respect to the series of wells when it passes over said series of wells.
A method according to any one of the claims 1 to 7, characterised in that said predetermined quantity ranges between 0.00025 and 100 µl.
A method according to any one of the claims 1 to 8, characterised in that said series of wells is included in an array of several series of wells, extending from one side of the array to the other, at least a number of said series of wells being filled with said predetermined quantity of fluid by passing said outlet over said number of series of wells.
A method according to claim 9, characterised in that after having added said predetermined quantity of fluid into each well of one of said series of wells, said fluid outlet is moved with respect to the array in an area outside said array to pass to another one of said number of series of wells.